Download Periaqueductal Gray Metabotropic Glutamate Receptor Subtype 7

J Neurophysiol 98: 43–53, 2007.
First published May 16, 2007; doi:10.1152/jn.00356.2007.
Periaqueductal Gray Metabotropic Glutamate Receptor Subtype 7 and 8
Mediate Opposite Effects on Amino Acid Release, Rostral Ventromedial
Medulla Cell Activities, and Thermal Nociception
Ida Marabese,1,* Francesca Rossi,1,* Enza Palazzo,1 Vito de Novellis,1 Katarzyna Starowicz,2 Luigia Cristino,3
Daniela Vita,1 Luisa Gatta,1 Francesca Guida,1 Vincenzo Di Marzo,2 Francesco Rossi,1 and Sabatino Maione1,2
1
Department of Experimental Medicine, Section of Pharmacology “L. Donatelli,” Faculty of Medicine and Surgery, Second University of
Naples, Naples; 2Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli,
Italy; and 3Endocannabinoid Research Group, Institute of Cybernetics “E. Caianiello”, Consiglio Nazionale delle Ricerche, Pozzuoli
(Naples), Italy
Submitted 29 March 2007; accepted in final form 8 May 2007
INTRODUCTION
It has been shown that metabotropic glutamate subtype 7 and
8 receptors (mGluR7 and mGluR8), similarly to mGluR4 and
mGluR6, the other group III mGluR subtypes, function as
presynaptic receptors that modulate glutamate and GABA
*
I. Marabese and F. Rossi contributed equally to this work.
Address for reprint requests and other correspondence: S. Maione, Dept. of
Experimental Medicine, Sect. of Pharmacology “L. Donatelli,” Faculty of
Medicine and Surgery, Second University of Naples, Via Constantinopoli,
Naples 16 80138, Italy (E-mail sabatino.maione@unina2.it).
www.jn.org
releases (Cartmell and Schoepp 2000; Schaffhauser et al. 1998;
Schoepp 2001). Presynaptic modulation of the release of these
amino acids at spinal and periaqueductal gray (PAG) levels
may be effective in alleviating pain (Marabese et al. 2006;
Thomas et al. 2001). Indeed, the PAG is an important processing center for the descending control of nociception (Harris
1996), and glutamate and GABA play a critical role in processing pain at this level (Behbehani and Fields 1979; Harris
and Hendrickson 1987; Millan et al. 1987; Moreau and Fields
1986). We have previously shown that mGluRs modulate
glutamate, GABA, and glycine releases within the PAG (de
Novellis et al. 2002, 2003), contributing to the tonic modulation of nociception (Berrino et al. 2001; Maione et al. 2000).
More recently, we have found that presynaptic mGluR8 are
expressed within the PAG on both GABAergic and glutamatergic neurons, and their stimulation leads to a facilitation of
glutamate and an inhibition of GABA releases in rats (Marabese et al. 2005). Because GABAergic interneurons tonically
inhibit the PAG antinociceptive pathway (Moreau and Fields
1986), the mGluR8 stimulation-induced increase in glutamate
and reduction in GABA at that level may be crucial to produce
analgesia. Indeed, stimulation of mGluR8 within the PAG has
an important antinociceptive effect in inflammatory and neuropathic pain in mice (Marabese et al. 2006). Conversely, PAG
mGluR7, unlike mGluR8, subtype facilitates pain transmission
in mice (Marabese et al. 2006). It is reasonable to suppose that
the opposite effects induced by PAG mGluR4/8 and mGluR7 on
pain might be due to the preferential location of the mGluR7 on
glutamatergic synapses (Bradley et al. 1996; Shigemoto et al.
1997). Thus their possible main autoreceptor role on glutamate
terminals may justify an inhibition of the excitatory output in
the PAG antinociceptive pathway and consequently the appearance of the mGluR7-induced hyperalgesia. In this study, we
decided to further verify our hypothesis (the opposite effect of
mGluR7 and mGluR8 on pain modulation) by performing in
vivo microdialysis, behavioral, and electrophysiological studies. Thus the effects of PAG mGluR7 stimulation on glutamate
and GABA release and of mGluR7 and mGluR8 on rostral
ventromedial medulla (RVM) cell activities and tail flickrelated behavioral and electrophysiological responses have
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0022-3077/07 $8.00 Copyright © 2007 The American Physiological Society
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Marabese I, Rossi F, Palazzo E, de Novellis V, Starowicz K,
Cristino L, Vita D, Gatta L, Guida F, Di Marzo V, Rossi F,
Maione S. Periaqueductal gray metabotropic glutamate receptor
subtype 7 and 8 mediate opposite effects on amino acid release,
rostral ventromedial medulla cell activities, and thermal
nociception. J Neurophysiol 98: 43–53, 2007. First published May
16, 2007; doi:10.1152/jn.00356.2007. The current study has investigated the involvement of periaqueductal gray (PAG) metabotropic glutamate subtype 7 and 8 receptors (mGluR7 and mGluR8)
in modulating rostral ventromedial medulla (RVM) ongoing and
tail flick–related ON and OFF cell activities. Our study has also
investigated the role of PAG mGluR7 on thermoceptive threshold
and PAG glutamate and GABA release. Intra-ventrolateral PAG
(S)-3,4-dicarboxyphenylglycine [(S)-3,4-DCPG (2 and 4 nmol/rat)]
or N,N I -dibenzhydrylethane-1,2-diamin dihydrochloride
(AMN082, (1 and 2 nmol/rat), selective mGluR8 and mGluR7
agonists, respectively, caused opposite effects on the ongoing
RVM ON and OFF cell activities. Tail flick latency was increased or
decreased by (S)-3,4-DCPG or AMN082 (2 nmol/rat), respectively.
(S)-3,4-DCPG reduced the pause and delayed the onset of the OFF
cell pause. Conversely, AMN082 increased the pause and shortened the onset of OFF cell pause. (S)-3,4-DCPG or AMN082 did not
change the tail flick-induced onset of ON-cell peak firing. The tail
flick latency and its related electrophysiological effects induced by
(S)-3,4-DCPG or AMN082 were prevented by (RS)-␣-methylserine-o-phosphate (100 nmol/rat), a group III mGluR antagonist.
Intra-ventrolateral PAG perfusion with AMN082 (10 and 25 ␮M),
decreased thermoceptive thresholds and glutamate extracellular
levels. A decrease in GABA release was also observed. These
results show that stimulation of PAG mGluR8 or mGluR7 could
either relieve or worsen pain perception. The opposite effects on
pain behavior correlate with the opposite roles played by mGluR7
and mGluR8 on glutamate and GABA release and the ongoing and
tail flick-related activities of the RVM ON and OFF cells.
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METHODS
Animals
Male Wistar rats (250 –300 g) were housed three per cage under
controlled illumination (12:12 h light:dark cycle; light on 06.00 h) and
environmental conditions (ambient temperature: 20 –22°C, humidity:
55– 60%) for ⱖ1 wk before the commencement of experiments. Rat
chow and tap water were available ad libitum. The experimental
procedures were approved by the Animal Ethics Commitee of the
Second University of Naples. Animal care was in compliance with
Italian (D.L. 116/92) and European Economic Committee (O.J. of
E.C. L358/1 18/12/86) regulations on the protection of laboratory
animals. All efforts were made to minimize animal suffering and to
reduce the number of animals used.
Surgical preparation for intra-PAG microinjections
To perform direct intra-ventrolateral PAG administrations of drugs
or respective vehicle, artificial cerebrospinal fluid [ACSF, composition (in mM): 2.5 KCl, 125 NaCl, 1.18 MgCl2, and 1.26 CaCl2], rats
were anesthetized with pentobarbital (60 mg/kg ip), and a 23-gauge,
12 mm-long stainless steel guide cannula was stereotaxically lowered
until its tip was 1.5 mm above the ventrolateral PAG by applying
coordinates from the atlas of Paxinos and Watson (1986) (A: ⫺7.8
mm and L: 0.5 mm from bregma, V: 4.3 mm below the dura).
Ventrolateral PAG was considered in this study because we have
performed previous studies in the same area (Maione et al. 2006;
Marabese et al. 2006). The cannula was anchored with dental cement
to a stainless steel screw in the skull. We used a David Kopf
stereotaxic apparatus (David Kopf Instruments, Tujunga, CA) with
the animal positioned on a homeothermic temperature control blanket
(Harvard Apparatus Limited, Edenbridge, Kent, UK). The guide
cannula for intra-PAG microinjection was implanted on the same day
as the electrophysiological and tail flick latency recordings. Direct
intra-ventrolateral PAG administration of drugs or respective vehicle
was conducted with a stainless steel cannula connected by a polyethylene tube to a SGE 1-␮l 26-gauge syringe, inserted through the guide
cannula and extended 1.5 mm beyond the tip of the guide cannula to
reach the ventrolateral PAG. Volumes of 200 nl drug solutions, or
vehicle, were injected into the ventrolateral PAG over a period of 60 s,
and the injection cannula was gently removed 2 min later. At the end
of the experiment, a volume of 200 nl of neutral red (0.1%) was also
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injected in the ventrolateral PAG 30 – 40 min before killing the rat.
Rats were then perfused intracardially with 20 ml phosphate buffer
solution (PBS) followed by 20 ml 10% formalin solution in PBS. The
brains were removed and immersed in a saturated formalin solution
for 2 days. The injection site was ascertained by using two consecutive sections (40 ␮m), one stained with cresyl violet to identify nuclei
and the other unstained to determine dye spreading. Only those rats
the microinjected site of which was located within the ventrolateral
PAG were used for data computation.
RVM extracellular recording and tail flick test
After implantation of the guide cannula into the ventrolateral PAG,
a tungsten microelectrode was stereotaxically (Paxinos and Watson
1986) lowered through a small craniotomy into the RVM to record the
activity of neutral, ON, and OFF cells. As first described by Fields et al.
(1983), these neurons were identified by their responses to thermal
noxious stimulus. OFF cells were inhibited, ON cells excited, and
neutral cells unaffected by application of a thermoceptive stimulation
of the tail able to evoke a withdrawal (tail flick responses) (Fields et
al. 1983; Leung and Mason 1999; Mason 2005). ON and OFF cells also
responded (a sudden increase or a decrease in the firing rate, respectively) to noxious stimulation at the hind paws (press/pinch) or to light
touch of the corneal surface. In few cases, once RVM neutral, ON, and
OFF cells were identified, we also performed intra-PAG morphine
microinjections (2 ␮g/rat, 200 nl) to further verify their physiological
characteristics (data not included in this study). Neutral cells did not
modify their spontaneous activity after application of drugs or different nociceptive stimuli.
Anesthesia was maintained with a constant, continuous infusion of
propofol (5–10 mg 䡠 kg⫺1 䡠 h⫺1 iv). Anesthesia was adjusted so that
tail flicks were elicited with a constant latency of 4 –5 s. A thermal
stimulus was elicited by a radiant heat source of a tail flick unit (Ugo
Basile, Varese, Italy) focused on the rat tail ⬃3–5 cm from the tip.
From 35°C, the temperature increased linearly to 53°C and was
adjusted at the beginning of each experiment to elicit a constant tail
flick latency. Tail flicks were elicited every 3– 4 min for ⱖ15–20 min
prior to microinjecting drugs, or respective vehicle, into the PAG.
Extracellular single-unit recordings were made in the RVM with
glass-insulated tungsten filament electrodes (3–5 M⍀; FHC Frederick
Haer) using the following stereotaxic coordinates: 2.8 –3.3 mm caudal
to lambda, 0.04 – 0.9 mm lateral, and 8.9 –10.9 mm depth from the
surface of the brain (Paxinos and Watson 1986). The recorded signals
were amplified and displayed on analog and digital storage oscilloscope to ensure that the unit under study was unambiguously discriminated throughout the experiment. Signals were also fed into a window
discriminator the output of which was processed by an interface (CED
1401; Cambridge Electronic Design) connected to a Pentium III PC.
Spike2 software (CED, version 4) was used to create peristimulus rate
histograms on-line and to store and analyze digital records of singleunit activity off-line. Configuration, shape, and height of the recorded
action potentials were monitored and recorded continuously using a
window discriminator and Spike2 software for on- and off-line analysis. Once cells were identified from their background activities, we
optimized spike size before all treatments. This study only included
neurons the spike configuration of which remained constant and could
clearly be discriminated from activity in the background throughout
the experiment, indicating that the activity from one neuron only and
from that same neuron was measured. Only one neuron was recorded
in each rat.
Microdialysis procedure
Intra-PAG microdialysis experiments were performed in awake and
freely moving rats. In brief, rats were anesthetized with pentobarbital
(60 mg/kg ip) and stereotaxically implanted with concentric microdialysis probes, which were constructed as previously described (Biggs
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been evaluated in the current study. Three neuronal classes
with distinct physiology and pharmacology are found in the
RVM (Fields et al. 1991). Cells of one class, “neutral cells,”
show no modification in spontaneous activity associated with
nociceptive stimulation. On the other hand, there is evidence
that as regards the other two classes of cells, the “ON cells” and
the “OFF cells,” have specific roles in nociceptive modulation.
ON cells show a burst of activity just prior to withdrawal
reflexes, and OFF cells are inhibited just prior to withdrawal
reflexes. These cells respond in the opposite way to pharmacological stimulation with opioid receptor agonists: systemic or
local injections of opioid receptor agonists sufficient to inhibit
nociceptive reflexes inhibit ON-cell and increase OFF-cell activities (Fields et al. 1983; Heinricher and Tortorici 1994). A
greater understanding of the role of mGluR7 and mGluR8
within the PAG-RVM circuitry in the modulation of two
functionally counteracting neurotransmitters, such as glutamate and GABA, and in the electrophysiological and behavioral pain responses might provide further insight into the
pathophysiology of pain and new approaches to its pharmacological control.
PAG mGluRs IN PAIN CONTROL
Thermal withdrawal latency during microdialysis
Thermal nociception was evaluated by using Plantar Test Apparatus (Ugo Basile, Varese, Italy). On the day of the experiment, each
animal, which had been previously implanted with a microdialysis
probe, was placed in a plastic cage (22 ⫻ 17 ⫻ 14 cm; length ⫻
width ⫻ height) with a glass floor. After a 30-min habituation period,
the plantar surface of the hind paw was exposed to a beam of radiant
heat through the glass floor within the time interval between dialysate
sample collection. The radiant heat source consisted of an infrared
bulb (Osram halogen-bellaphot bulb; 8 V, 50 W). A photoelectric cell
detected light reflected from the paw and turned off the lamp when
paw movement interrupted the reflected light. Paw withdrawal latency
was automatically displayed to the nearest 0.1 s; the cut-off time was
20 s to prevent tissue damage. Vehicle or drug perfusion by reverse
microdialysis was performed after the collection of five basal microdialysis samples and the simultaneous recording of five basal thermal
withdrawal latencies every 30 min. Thermoceptive responses were
expressed as paw withdrawal latency in seconds (means ⫾ SE).
Nociceptive responses were measured every 30 min for a period of 5 h
and 30 min.
Immunohistochemistry
Animals were killed (pentobarbital, 60 mg/kg ip) and perfused
transcardially with saline followed by ice-cold 4% paraformaldehyde
in 0.1 M phosphate buffer (PB), pH 7.4. Brains were removed and
paraffin embedded. Microtome sections were cut at a thickness of 8
␮m, collected on slides in three series each consisting of pairs of serial
sections mounted onto gelatin-coated slides (Mezel). For mGluR7 and
mGluR8 antigen immunohistochemistry, the sections were dewaxed
and rehydrated, immersed in diluted Antigen Unmasking Solution
(Vector Laboratories, Burlingame, CA), and then proceeded for ABC
immunohistochemistry technique. The sections were reacted for 10
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min in 0.3% H2O2 to inactivate endogenous peroxidase activity and
incubated for 1 h at room temperature in 10% normal goat serum
(NGS, Vector Laboratories) in 0.1 M Tris-HCl-buffered saline, pH 7.3
(TBS), containing 0.3% Triton X-100 and 0.05% sodium azide
(Sigma-Aldrich). The sections were then incubated for 24 h at 4°C
with rabbit polyclonal mGluR7 antibody (Abcam, Cambridge, UK)
diluted at 1:200 in NGS or guinea pig polyclonal mGluR8 antibody
(Chemicon International). After three rinses, the sections were incubated for 2 h in biotinylated goat anti-rabbit diluted 1:100 in NGS or
biotinylated goat anti-guinea pig IgGs diluted 1:500 in NGS (both
Vector Laboratories), followed by incubation for 1 h at room temperature in the avidin-biotin-peroxidase solution (ABC Kit; Vectastain,
Vector) in TBS, and then in 0.05‰ 3–3⬘diaminobenzidine (DAB
Sigma Fast, Sigma-Aldrich) in 0.01 M TBS. Then the brain sections
were washed in water, and all sections were dehydrated in alcohol,
cleared in xylene, and mounted in dibutylpthalate polystyrene xylene
(DPX; Merck). Controls included: preabsorption of diluted antibody
with respective immunizing peptide (synthesized on custom request
by Inbios) and omission of either the primary antisera or the secondary antibodies. These control experiments did not show staining. The
sections were investigated under bright-filed illumination (Leica DM
IRB microscope). Images were acquired using the digital camera
Leica DFC 320 connected to the microscope and the image analysis
software Leica IM500. Digital images were processed in Adobe
Photoshop with brightness and contrast being the only adjustments
made.
Treatments
Groups of 8 –10 animals per treatment were used with each animal
being used for one treatment only.
For in vivo extracellular recording and tail-flick test, rats receiving
intra-ventrolateral PAG administration of vehicle or different doses of
(S)-3,4-dicarboxyphenylglycine [(S)-3,4-DCPG] or N,NI-dibenzhydrylethane-1,2-diamin dihydrochloride (AMN082), alone or in combination with (RS)-␣-methylserine-o-phosphate (MSOP), were
grouped as follows. 1) A group of rats was implanted with guide
cannulae and received an intra-ventrolateral PAG microinjection of 50
nl of ACSF and served as a control of the intra-ventrolateral PAG
drug microinjection. 2) Groups of rats received intra-ventrolateral
PAG administration of (S)-3,4-DCPG (2 and 4 nmol/rat) and MSOP
(100 nmol/rat) alone or (S)-3,4-DCPG (4 nmol/rat) in combination
with MSOP (100 nmol/rat). When (S)-3,4-DCPG was administered in
combination with MSOP, the latter was centrally delivered 3 min
before the administration of (S)-3,4-DCPG. 3) Groups of rats received
intra-ventrolateral PAG administration of AMN082 (1 and 2 nmol/rat)
alone or in combination with intra-ventrolateral PAG MSOP (100
nmol/rat). When AMN082 (2 nmol/rat) was administered in combination with MSOP, the latter was centrally delivered 3 min before the
administration of AMN082.
For the combined plantar test and microdialysis experiments, rats
receiving intra-ventrolateral PAG perfusion with vehicle or different
concentrations of AMN082, alone or in combination with MSOP,
were grouped as follows. 1) Groups of rats implanted with a concentric microdialysis probe into the ventrolateral PAG were perfused for
30 min with ACSF or Ca2⫹-free ACSF or TTX (1 ␮M) and served as
a control of the intra-ventrolateral PAG drug perfusion, or to establish
the origin of GABA and glutamate releases. 2) Groups of rats received
intra-ventrolateral PAG perfusion with AMN082 (10 and 25 ␮M),
MSOP (0.5 mM) alone, or AMN082 (25 ␮M) in combination with
MSOP (0.5 mM).
In agreement with our previous microdialysis experiments, we used
higher agonist concentrations compared with their in vitro EC/IC50
because of the relatively low probe recovery (⬃20%), the more
efficient uptake/metabolism by glial and neural cells in vivo, and the
drug diffusion from the probe site. Nevertheless, we have previously
found (Marabese et al. 2006) and confirmed here that the antagonist
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et al. 1992), into the ventrolateral PAG using coordinates: A: ⫺7.5
mm and L: ⫹0.5 mm from bregma, V: 6.5 mm below the dura
(Paxinos and Watson 1986). After a postoperative recovery period of
⬃18 h, dialysis was commenced by perfusing ACSF at a rate of 0.8
␮l/min using a Harvard Apparatus infusion pump (mod. 22). The
Ca2⫹-free ACSF was produced from normal ACSF by omitting Ca2⫹
(composition, in mM: 2.5 KCl, 125 NaCl, 1.18 MgCl2). On the day of
the experiment, each animal was placed in a Plexiglas cage and
allowed to move freely. After an initial 60-min equilibration period,
12 consecutive 30-min dialysate samples were collected. Rats received Ca2⫹-free ACSF, TTX, or all other drugs through the microdialysis probe during a period of 30 min (from 120 to 150 min from
the commencement of the microdialysis sample collection). On completion of each experiment, rats were anesthetized with pentobarbital,
and their brains were perfused-fixed via the left cardiac ventricle with
heparinized paraformaldehyde saline (4%). Brains were removed 120
min after fixation, and coronal sections cut to verify probe placements.
Dialysates were analyzed for amino acid content using an HPLC
method. The system comprised two Gilson pumps (model No. 303), a
C18 reverse-phase column, a Gilson refrigerated autoinjector (model
No. 231), and a Gilson fluorimetric detector (model No. 121). Dialysates were precolumn derivatized with o-pthaldialdehyde (OPA) (10
␮l dialysate ⫹10 ␮l OPA), and amino acid conjugates were resolved
using a gradient separation. The detection limit of GABA and glutamate in 10-␮l samples was ⬃0.5–1 and 2–3 pmol, respectively. The
mobile phase consisted of two components: 1) 50 mM sodium
dihydrogen orthophosphate, pH 5.5, with 20% methanol and 2) 100%
methanol. Gradient composition was determined with an Apple microcomputer installed with Gilson gradient management software.
The mobile phase flow rate was maintained at 1.0 ml/min. Data were
collected using a Dell Corporation PC system 310 interfaced to the
detector via a Drew data-collection unit.
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MSOP was effective already at 500 ␮M, a fairly low concentration as
compared with its IC50 in vitro (Thomas et al. 1996), in erasing the
effect of AMN082 or (S)-3,4-DCPG. Indeed this may be the consequence of some limitations of this technique in vivo, as there is no
way to make any definitive assumption on the final concentration of
drug reaching a specific target (de Lange et al. 1997).
Drugs
(S)-3,4-DCPG, AMN082, and (RS)-␣-methylserine-o-phosphate,
MSOP, were purchased from Tocris Cookson, Bristol, UK. Tetrodotoxin, TTX, was purchased from Sigma. TTX, (S)-3,4-DCPG, MSOP,
and AMN082 were dissolved in ACSF with final pH ⫽ 7.2 for
intra-PAG microinjection or perfusion.
Single-unit extracellular recording (action potentials) was analyzed
off-line from peristimulus rate histograms using Spike2 software
(CED, version 4). The neuron responses, before and after intraventrolateral PAG vehicle or drug microinjections, were measured
and expressed as spike/second (Hz). Baseline activities of neurons
were measured between tail flicks. In particular, basal values were
obtained by averaging the activities recorded 30 –50 s before the
application of three to four thermal stimulations (each stimulation trial
was performed every 3– 4 min). Data are presented as means ⫾ SE
either of changes in time latencies (tail flick test) or changes in neuron
responses (extracellular recordings). Statistical comparisons of values
from different treated groups of rats were made using the two-way
ANOVA for repeated measures followed by the Tukey/Kramer test
for post hoc comparisons.
To analyze tail flick-related ON cell activities (before and after drug
treatment), the ongoing activity (spike/s) was determined 40 –50 s
before tail flick application, and then the peak of ON cell activity
related to the tail flick (peak firing) was quantified. Interspike interval
was used to distinguish between the beginning of an ON cell burst and
ongoing activity. Tail flick-related ON cell firing was calculated as the
number of spikes in the 2-s interval beginning 0.5 s before the tail
flick. Comparisons between pre- and posttreatment ongoing activity
and tail flick-related cell burst were performed by applying the
nonparametric Wilcoxon matched-pairs signed-rank test. Furthermore, we calculated the ON cell burst latency; that is, the interval
between the onset of the applied noxious radiant heat and the beginning of the tail flick-related cell burst. Burst latency was analyzed
using two-way ANOVA for repeated measures followed by the
Tukey-Kramer test for post hoc comparisons.
We also performed analysis of tail flick related OFF cell activities
before and after drug treatments. The ongoing activity (spike/s, 40 –50
s before radial heat application), the latency to onset of the OFF cell
pause (time between the onset of heat application and the last action
potential prior to the tail flick), and the duration of the cell pause (the
interval between the pause onset and the 1st spike after the tail flick)
were determined. Comparisons between pre- and post-treatment ongoing activity and cell pause related to tail flick were performed by
applying the nonparametric Wilcoxon matched-pairs signed-rank test.
The latency to the onset of the cell pause was analyzed with a
two-way ANOVA for repeated measures with the Tukey-Kramer test
for post hoc comparisons.
Microdialysis, tail flick or plantar test data are represented as
means ⫾ SE, and statistical analysis of these data were performed by
two-way ANOVA for repeated measures followed by the StudentNewman-Keuls multiple comparisons test to determine the statistical
significance between different treated groups of rats. Differences were
considered significant at P ⬍ 0.05.
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Effect of (S)-3,4-DCPG or AMN082 on the ongoing activities
of RVM neutral, on, and off cells
The results are based on RVM neurons (group size ⫽ 8 –10;
1 cell recorded from each animal per treatment) at a depth of
8, 905–10,910 ␮m from the surface of the brain, the estimated
location of the neurons being in nucleus raphe magnus, nucleus
reticularis gigantocellularis pars ␣, and nucleus reticularis
paragigantocellularis. All recorded neurons were spontaneously active and discharged with a mean frequency of 7.7 ⫾
0.4 (neutral cells), 6.7 ⫾ 0.5 (ON cells) and 8.0 ⫾ 0.4 (OFF cells)
spike/s. These neurons were identified by the characteristic OFF
cell pause and ON cell burst of activity just before tail flick
responses. Neutral cells did not modify their spontaneous
activity during or after application of thermoceptive stimulation of the tail. Microinjections of (S)-3,4-DCPG (2 and 4
nmol/rat) into the ventrolateral PAG caused a dose-dependent
decrease in the firing activity of the ON cells, which was
significant between 3 and 15 min, and maximal 12 min after
administration of the highest dose (Figs. 1A and 2A). The same
treatment produced a very rapid increase in the firing activity
of the OFF cells, which was already significant after 3 min and
maximal after 9 min from administration of the highest dose
(Figs. 1B and 2B). Unlike (S)-3,4-DCPG, microinjections of
AMN082 (1 and 2 nmol/rat) into the ventrolateral PAG caused
a dose-dependent increase in the firing activity of the ON cells,
which was significant between 3 and 24 min and maximal 12
min after administration (Figs. 1C and 2C), and a very rapid
decrease in the firing activity of the OFF cells, already significant after 3 min and maximal 9 min after administration of the
highest dose (Figs. 1D and 2D). The effects of (S)-3,4-DCPG
or AMN082 were prevented by pretreatment with MSOP (100
nmol/rat). MSOP (100 nmol/rat) did not significantly change
per se the RVM ON and OFF cell ongoing activities (Fig. 1).
Spontaneous activities of RVM neutral neurons (n ⫽ 5) as
identified by their nonresponsiveness to tail flick were also
analyzed before and after microinjections of (S)-3,4-DCPG (4
nmol/rat) or AMN082 (2 nmol/rat) into the ventrolateral PAG.
Both drug treatments failed to cause any change in their
spontaneous activities (Fig. 2, E and F).
Effect of (S)-3,4-DCPG and AMN082 on tail flick-related
changes on RVM cell activities
The highest doses of (S)-3,4-DCPG (4 nmol/rat) or
AMN082 (2 nmol/rat) modified tail flick-related OFF cell activity. (S)-3,4-DCPG (4 nmol/rat) did not significantly change
the ON cell onset of burst (4.5 ⫾ 0.7 vs. 6.7 ⫾ 0.9 s), whereas
it decreased the duration of the OFF cell pause (from 10.5 ⫾ 3.2
to 4.2 ⫾ 2.2 s; P ⬍ 0.05; Figs. 2, A and B, and 3A).
(S)-3,4-DCPG did not significantly affect tail flick-induced
ON-cell peak firing (from 16.5 ⫾ 3.7 to 11.2 ⫾ 4.2 spike/s),
whereas it delayed the onset of OFF cell pause (from 4.4 ⫾ 0.4
to 9.3 ⫾ 0.3 s; P ⬍ 0.05; Figs. 2, A and B, and 3A). AMN082
(2 nmol/rat) did not significantly change the ON cell onset of
burst (4.5 ⫾ 0.7 vs. 3.8 ⫾ 0.6 s), but it increased the duration
of the OFF cell pause (from 10.5 ⫾ 3.4 to 18.4 ⫾ 3.2 s; P ⬍
0.05; Figs. 2, C and D, and 3B). AMN082 did not significantly
affect tail flick-induced ON cell peak firing (from 17.5 ⫾ 3.4 to
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Statistics
RESULTS
PAG mGluRs IN PAIN CONTROL
19.7 ⫾ 5.2 spike/s) either, but it did shorten the onset of OFF
cell pause (from 4.5 ⫾ 0.5 to 2.2 ⫾ 0.3 s; P ⬍ 0.05; Figs. 2,
C and D, and 3B). Unlike RVM ON and OFF cells, the administration of (S)-3,4-DCPG or AMN082 did not affect the
activity of neutral cells before and after thermoceptive stimulation (Fig. 2, E and F).
Effect of (S)-3,4-DCPG and AMN082 on tail-flick latencies
A
°
6
° °
* * *
*
* *
3
6
9
*
*
*
*
*
*
*
6
°
*
*
°
°
*
°
° °
°
° °
°
*
3
9 12 15 18 21 24 27 30 33 36 39min
3
6
9 12 15 18 21 24 27 30 33 36 39 min
D
B
OFF CELLS
*
9
°
6
*
*
°
*
ACSF
(S)-3,4-DCPG 2 nmol
(S)-3,4-DCPG 4 nmol
MSOP 100 nmol +(S)-3,4-DCPG
MSOP 100 nmol
*
*
°
°
*
12
Spikes/sec
12
Spikes/sec
ACSF
AMN082 1 nmol
AMN082 2 nmol
MSOP 100 nmol +AM
MSOP 100 nmol
ON CELLS
*
*
*
3
Intra-ventrolateral PAG perfusion with the selective mGluR7
agonist, AMN082 (10 and 25 ␮M), decreased the extracellular
12
9
° ° °
Effect of AMN082 on PAG glutamate and GABA
extracellular values
C
ACSF
(S)-3,4-DCPG 2 nmol
(S)-3,4-DCPG 4 nmol
MSOP 100 nmol +(S)-3,4-DCPG
MSOP 100 nmol
ON CELLS
The mean basal extracellular GABA, glutamate and glutamine levels in the PAG (not corrected for probe recovery of
24 ⫾ 8, 30 ⫾ 6, and 35 ⫾ 7% for GABA, glutamate and
glutamine, respectively) were 6.9 ⫾ 0.5, 25 ⫾ 5, and 458 ⫾ 29
pmol/10 ␮l of dialysate (means ⫾ SE), respectively. These
values are concordant with those obtained in our previous
studies and with those of other laboratories (Marabese et al.
2005; Maione et al. 1999, 2000; Renno et al. 1992). Each
animal was used only once, and the reported basal values of
glutamate, GABA, and glutamine are the mean concentrations
obtained from all experiments pooled as controls. Intra-PAG
perfusion with TTX (1 ␮M) decreased the extracellular levels
of glutamate and GABA (58 ⫾ 5 and 51 ⫾ 7% of basal value,
respectively; Fig. 5, A and B). Similarly, Ca2⫹ free ACSF
decreased the extracellular levels of glutamate and GABA
(45 ⫾ 5 and 47 ⫾ 6% of basal value, respectively; Fig. 5, A and
B).
Spikes/sec
Spikes/sec
12
Effect of TTX and Ca2⫹-free ACSF on PAG glutamate and
GABA extracellular values
°
ACSF
AMN082 1 nmol
AMN082 2 nmol
MSOP 100 nmol +AMN082
MSOP 100 nmol
OFF CELLS
°
9
3
6
9
12 15 18 21 24 27 30 33 36 39 min
°
*
6
*
3
°
°
* *
°
*
*
*
*
*
21
24
27
*
°
°
*
*
3
3
6
9
12
FIG.
15
18
30 33
36
39 m in
1. Effect of artificial cerebrospinal fluid (ACSF), (S)-3,4-dicarboxyphenylglycine [(S)-3,4-DCPG], or N,N⬘-dibenzhydrylethane-1,2-diamin dihydrochloride (AMN082) on the spontaneous firing of rostral ventromedial medulla (RVM) ON (A and C) or OFF (B and D) cells. A and B: effect of intra-ventrolateral
periaqueductal gray (PAG) administration of (S)-3,4-DCPG (2 and 4 nmol/rat) and (RS)-␣-methylserine-o-phosphate (MSOP, 100 nmol/rat) alone or
(S)-3,4-DCPG (4 nmol/rat) in combination with MSOP (100 nmol/rat). C and D: effect of AMN082 (1 and 2 nmol/rat) and MSOP (100 nmol/rat) alone or
AMN082 (2 nmol/rat) in combination with MSOP (100 nmol/rat). 1, drug microinjections. Each point represents the mean ⫾ SE of 8 –10 neurons. *, significant
differences vs. ACSF and E significant differences vs. (S)-3,4-DCPG (4 nmol/rat) or AMN082 (2 nmol/rat). P values ⬍0.05 were considered statistically
significant.
J Neurophysiol • VOL
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Tail flicks were elicited every 3– 4 min for ⱖ20 min prior to
microinjecting drugs, or respective vehicle, into the ventrolateral PAG. Data related to pretreatment interval were considered as basal tail flick latencies (4.5 ⫾ 0.3 s). Intra-ventrolateral PAG microinjection of vehicle did not change the tail flick
latency as compared with basal values (4.6 ⫾ 0.5 s; Fig. 4).
Tail flick latency was increased to 7.3 ⫾ 0.6 and 10.2 ⫾ 0.8 s
or decreased to 2.9 ⫾ 0.3 and 1.7 ⫾ 0.6 s by intra-ventrolateral
PAG microinjections of (S)-3,4-DCPG (2 and 4 nmol/rat) or
AMN082 (1 and 2 nmol/rat), respectively (P ⬍ 0.05; Fig. 4, A
and B). The effects of the higher dose of (S)-3,4-DCPG or
AMN082 were prevented by pretreatment with MSOP (100
nmol/rat; Fig. 4, A and B). MSOP (100 nmol/rat) did not
significantly change per se the tail flick latencies (Fig. 4).
47
48
MARABESE ET AL.
Tail Flick Latency (s)
A
ACSF
(S)-3,4-DCPG 2 nmol
(S)-3,4-DCPG 4 nmol
MSOP 100 nmol+(S)-3,4-DCPG
MSOP 100 nmol
12
*
*
10
*
*
8
*
6
°
°
4
2
*
*
°
°
°
45
60
*
0
5
pre-drug
15
30
post-drug
min
Tail Flick Latency (s)
ACSF
AMN082 1 nmol
AMN082 2 nmol
MSOP 100 nmol +AMN082
MSOP 100 nmol
10
8
6
°
4
*
*
0
5
pre-drug
2. Examples of ratemeter records that illustrate the effects of intraPAG microinjections of (S)-3,4-DCPG (4 nmol/rat; A and B) or AMN082 (2
nmol/rat; C and D) on either the ongoing or tail flick-related discharges of
identified RVM ON (A and C) and OFF (B and D) cells. Spontaneous activity of
RVM neutral cells were also analyzed before and after (S)-3,4-DCPG (4
nmol/rat; E) or AMN082 (2 nmol/rat; F) intra-PAG microinjections. Traces
report overall firing before and after drug injections into the ventrolateral PAG.
Πtail flick trials, 1-s bins. 1, time of microinjections within the ventrolateral
PAG. Scale bar 4 min.
°
°
*
*
*
2
FIG.
°
*
15
30
45
60
min
post-drug
FIG.
4. Tail flick latencies before and after microinjections into the ventrolateral PAG of ACSF, (S)-3,4-DCPG (2 and 4 nmol/rat), MSOP (100
nmol/rat) alone, or (S)-3,4-DCPG (4 nmol/rat) in combination with MSOP
(100 nmol/rat; A) and ACSF, AMN082 (1 and 2 nmol/rat), MSOP (100
nmol/rat) alone, or AMN082 (2 nmol/rat) in combination with MSOP (100
nmol/rat; B). Each point represents the mean ⫾ SE of 8 –10 observations. *,
significant differences vs. ACSF; E, significant differences vs. (S)-3,4-DCPG
(4 nmol/rat) or AMN082 (2 nmol/rat). P values ⬍0.05 were considered
statistically significant.
FIG. 3. Examples of ratemeter records with expanded time scale which illustrate the effects of intra-PAG microinjections of (S)-3,4-DCPG (4 nmol/rat) (A)
or AMN082 (2 nmol/rat) (B) on either the ongoing or tail flick-related discharge pauses of identified RVM OFF cells. Traces (all 3 traces from the same cell)
report overall firing before and 10 min after drug injections into the ventrolateral PAG. Œ, tail flick trials, 1-s bins. Scale bar, 0.5 min.
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B
PAG mGluRs IN PAIN CONTROL
A
150
100
Immunohistochemistry
*
*
50
*
*
*
*
*
0
0
30
60
90
120
150
180
min
210
240
270
300
330
B
ACSF
200
TTX 1 µM
Ca 2+ free
DISCUSSION
The involvement of group III mGluRs in modulating pain
responses has not yet been fully established (Fisher and
100
*
*
*
A
*
*
*
*
50
*
*
*
200
*
0
0
30
60
90
ACSF
AMN082 10 µM
AMN082 25 µM
MSOP 0.5 m M+AMN082 25 µM
*
120 150 180 210 240 270 300 330
min
5. Effects of ACSF, TTX (1 ␮M), or Ca2⫹-free ACSF on ventrolateral
PAG dialysate levels of glutamate (A) and GABA (B). —, period of TTX or
Ca2⫹-free ACSF perfusion. Each point represents the mean ⫾ SE of amino
acid extracellular concentrations as a percentage of the basal values (8 –10 rats
per group). *, significant difference vs. ACSF. P values ⬍0.05 were considered
statistically significant.
FIG.
levels of glutamate in a concentration-dependent manner (60 ⫾
6 and 21 ⫾ 9% of basal value, respectively; Fig. 6A). AMN082
(10 ␮M) did not change dialysate GABA concentration (69 ⫾
5% of basal value; Fig. 6B). The higher dose of AMN082 (25
␮M) produced a significant delayed effect on GABA extracellular values (56 ⫾ 6% of basal value; Fig. 6B). MSOP (0.5
mM), a group III mGlu receptor antagonist, perfused in combination with AMN082 (25 ␮M), antagonized the effect induced by AMN082 on extracellular glutamate and GABA
releases (Fig. 6, A and B).
% basal glutamate
% basal GABA
150
150
°
°
°
100
*
°
°
°
*
*
270
300
°
*
*
*
50
*
*
*
0
0
30
60
90
120
150
180
210
240
330
min
B
ACSF
AMN082 10 µM
AMN082 25 µM
MSOP 0.5 m M+AMN082 25 µM
200
150
°
°
°
°
100
*
50
*
Effect of AMN082 on thermal withdrawal latency
(Plantar test)
*
*
0
Because we have recently already evaluated the effect of
(S)-3,4-DCPG on nocifensive behavior (Marabese et al. 2006),
we decided to focus on the current study on the effect of
AMN082 when administered into the PAG matter. Intra-PAG
perfusion with AMN082 (10 –25 ␮M), by reverse microdialysis, induced a dose-dependent decrease in thermal withdrawal
latency (7.5 ⫾ 0.5 and 5.6 ⫾ 0.4 s vs. 10.2 ⫾ 0.4 s; P ⬍ 0.05).
When MSOP (0.5 mM) was perfused in combination with
J Neurophysiol • VOL
0
30
60
90
120
150
180
210
240
270
300
330
min
FIG. 6. Effects of ACSF, AMN082 (10 and 25 ␮M) alone, or AMN082 (25
␮M) in combination with MSOP (0.5 mM) on ventrolateral PAG dialysate
levels of glutamate (A) and GABA (B). —, period of drug perfusion. Each point
represents the mean ⫾ SE of amino acid extracellular concentrations as a
percentage of the basal values (8 –10 rats per group). *, significant difference
vs. ACSF; E, vs. AMN082 (25 ␮M). P values ⬍0.05 were considered
statistically significant.
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Immunohistochemical localization of mGluR7 and mGluR8
in rat PAG was determined by ABC immunohistochemistry
technique. We found mGluR7 and mGluR8 positive neurons in
all the PAG area. However, a higher density of mGlu7 positive
profiles with strong immunoreactivity (ir) was observed within
the ventrolateral sub-region (Fig. 8A). Numerous neurons display also strong mGluR8 immunoreactivity (Fig. 8D). Higher
magnification of these profiles revealed mGlu7-ir and mGlu8-ir
in cytoplasm on cell bodies and neuronal processes (Fig. 8, B
and E).
*
% basal GABA
% basal glutamate
AMN082 (25 ␮M), it fully antagonized the pronociceptive
effect of AMN082 but did not change per se thermal withdrawal latencies (Fig. 7). No overt behavioral changes were
observed in this study after intra-PAG administration of all the
drugs used in freely moving not unanesthetized rats. Rats
remained alert and generally active throughout the experiment
(not shown).
ACSF
TTX 1 µM
Ca 2+ free
200
49
50
MARABESE ET AL.
Thermal withdrawal latency (s)
ACSF
AMN082 10 µM
AMN082 25 µM
MSOP 0.5 mM +AMN082
MSOP 0.5 mM
12
°
°
°
*
8
4
*
*
150
180
°
°
*
*
*
*
210
240
*
0
0
30
60
90
120
270
300
330
min
7. Effects of intra-ventrolateral PAG perfusion by reverse microdialysis of ACSF, AMN082 (10 and 25 ␮M) and MSOP (0.5 mM) alone, or
AMN082 (25 ␮M) in combination with MSOP (0.5 mM) on thermal withdrawal latency in s. —, period of drug perfusion. Each point represents the
mean ⫾ SE of 8 –10 animals per group. *, significant differences vs. the ACSF;
E, significant differences vs. AMN082 (25 ␮M). P values ⬍0.05 were
considered statistically significant.
Coderre 1996; Neugebauer et al. 2000). Our previous studies
reported some important discrepancies in the action of group
III receptor ligands in modulating pain. In particular, blockade
of these receptors at PAG level, a major component of the
descending pain modulatory system, either decreased or increased thermoceptive responses in the mouse or rat, respectively (Maione et al. 1998, 2000; Palazzo et al. 2001). More
recently, however, we have found that selective stimulation of
the mGluR8 decreases nociceptive behavior in different pain
models in the mouse (Marabese et al. 2006).
Unlike mGluR4/8, PAG mGluR7 stimulation may be responsible for generating hyperalgesia. Indeed, a preliminary study
showed that intra-PAG microinjection of AMN082, a selective
FIG. 8. Immunohistochemical localization of mGluR7 (A–C) and mGluR8 (D–F) receptors in rat ventrolateral PAG as determined by ABC immunohistochemistry technique. A and D: general view of mGluR7 (A) and mGluR8 (B) immunoreactive distribution. B and E: high magnification of respective boxed areas
of A and D. Note the dense mGluR7 and mGluR8 immunolabeling in cellular cytoplasm and processes. C and F: negative control of mGluR7 (C) and mGluR8
(F) immunolabeling in ventrolateral PAG treated by respective primary antibodies preadsorbed with corresponding blocking peptides. Note the lack of
immunostaining. Images are representative of 3 different experiments. Aq, lumen of aqueduct. Scale bar 90 ␮m, except for B and D ⫽ 30 ␮m.
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FIG.
mGluR7 agonist, induced dose-dependent thermal hyperalgesia
(Marabese et al. 2006). One reason for the opposing effects
obtained from mGluR4/8 and mGluR7 in pain control at the
midbrain PAG could be due to the localization of the mGluR7
on glutamatergic synapses. A possible main auto-receptor role
for these subtype receptors on glutamate terminals might justify the decrease in the excitatory output of the PAG antinociceptive pathway. However, in this study we have only had the
possibility to see several profiles with strong mGluR7 immunoreactivity especially within ventrolateral PAG. From these
data, unfortunately it is not possible to distinguish whether
these cells correspond to PAG output excitatory neurons impinging on the RVM OFF cell population. Further ultra-structural studies would clarify such a possibility. Regarding
mGluR8-ir, as compared with mGluR7, it appeared to be more
diffusely expressed in many profiles within the PAG matter,
and this finding seems to confirm our previous data that
mGluR8 are present on both symmetrical and asymmetrical
synapses (Marabese et al. 2005).
We performed in vivo microdialysis experiments to shed
more light on the possibility that mGluR7 in the PAG may
inhibit mainly the release of glutamate. Group III mGluRs are
“classically” associated with a reduction in both glutamate and
GABA (Cartmell and Schoepp 2000; Schoepp 2001). Nevertheless, intra-PAG perfusion with (S)-3,4-DCPG by reverse
microdialysis increased glutamate, whereas it reduced GABA
release (Marabese et al. 2005). The opposite (facilitatory or
inhibitory) effects of mGluR8 stimulation on glutamate and
GABA release could be due to the fact that group III mGluR
stimulation, by decreasing GABA, increases glutamate release.
Thus even if such a possibility is not detectable by microdialysis, we speculate that the effects on glutamate and GABA
releases are not simultaneous but rather that one is the consequence of the other. Nevertheless, these neurochemical
changes are consistent with evidence that mGluR8 are ex-
PAG mGluRs IN PAIN CONTROL
J Neurophysiol • VOL
of the extracellular environment and make it so different from
the in vitro preparations.
Together with the PAG, the nuclei of the RVM constitute
part of the endogenous antinociceptive pathway. Two classes
of neurons (i.e., ON and OFF cells) in the RVM control pain
conversely (Fields and Basbaum 1999). When microinjected
into the PAG, (S)-3,4-DCPG decreased the ongoing activity of
the pro-nociceptive ON cells and increased the ongoing activity
of the anti-nociceptive OFF cells in the RVM, unlike AMN082,
which increased the ongoing activity of the pro-nociceptive ON
cells and decreased the ongoing activity of the anti-nociceptive
OFF cells. Unlike ON and OFF cells, spontaneous activity of
neutral cells was not affected by the intra-ventrolateral PAG
microinjection of (S)-3,4-DCPG or AMN082.
Thus selective stimulation of PAG mGluR8 or mGluR7 may
have modified the activity of PAG output neurons and either
increased or decreased the input required to modulate the ON
and the OFF cell spontaneous activities. Because the ON cells
may facilitate nociception (Fields et al. 1991), a delayed or
shortened onset of the ON cell burst with either mGluR8 or
mGluR7 might be expected to be a critical event to the
occurrence of analgesia or hyperalgesia. However, such a
possibility does not seem consistent with the current study or
with another previous study showing that the ON cell burst can
even be completely inhibited without any consequence to tail
flick latency (Heinricher and McGaraughty 1998). It is possible
that ON cell firing provides a critical regulatory pro-nociceptive
output in persistent pain states (Wiertelak et al. 1997) or in
other conditions such as opioid withdrawal. ON cells but not
OFF cells increased their spontaneous activity in naloxoneprecipitated hyperalgesia (Bederson et al. 1990). Based on
these considerations, spontaneous activity patterns of these
cells are critical in determining nociceptive thresholds to any
given noxious stimulus. Consistent with the idea that ongoing
activity of both ON and OFF cells modulate nociceptive responsiveness, the latency of the nociceptive tail-flick reflex is
shorter during periods of increased ON cell activity (Foo and
Mason 2005; Heinricher et al. 1989; Jinks et al. 2004). Thus
the gain in pain transmission is constantly changing, increasing
during periods of ON cell activity and decreasing when OFF cells
are active (Mason 2005; Ramirez and Vanegas 1989).
Stimulation of PAG mGluR8 or mGluR7, respectively, delayed or shortened the onset of the OFF cell pause and, respectively, decreased or increased the duration of the pause of these
cells. These effects seem to be critical in generating RVMmediated analgesia or hyperalgesia (de Novellis et al. 2005;
Heinricher and Tortorici 1994; McGaraughty and Heinricher
2002).
This study provides initial evidence that a mGluR8 agonist in
the PAG, similarly to opioids or cannabinoids, is able to inhibit
GABAergic tone, which, in turn, is responsible for the disinhibition of PAG antinociceptive output neurons impinging on
OFF neurons in the RVM (Behbehani and Fields 1979; de
Novellis et al. 2005; Maione et al. 2006; Richteimpr and
Behbehani 1991). Because many GABAergic interneurons are
tonically active in the PAG (Moreau and Fields 1986), the
mGluR8-induced decrease in the GABAergic tone may be
responsible for the PAG output excitatory neuron disinhibition
and therefore for the antinociception. Moreover, another important finding of this study is that the mGluR7 in the PAG
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pressed on both symmetrical and asymmetrical synapses at that
level (Marabese et al. 2005) and with the activation of an
endogenous pain inhibitory system and consequent PAG-mediated analgesia (Marabese et al. 2006).
Further weight is given to this possibility here because
AMN082 induced dose-dependent thermal hyperalgesia in the
plantar test and, simultaneously, a massive decrease in PAG
glutamate in the current study. As far as GABA is concerned,
AMN082 produced a slight and delayed decrease in the same
rats. This latter finding further suggests that the main effect of
AMN082 may be consequence of a direct action of this drug on
the excitatory glutamatergic cells. The possible synaptic nature
of glutamate and GABA in the PAG dialysates seems in part
confirmed by the fact that either TTX or Ca2⫹-free ACSF
perfusions almost halved their extracellular concentrations.
This finding suggests that almost 45–55% of extracellular
glutamate and GABA we measured in the PAG may function
as neurotransmitters. Nevertheless, one has to be cautious
when using in vivo microdialysis as these experiments do not
provide definitive results on synaptic release of either glutamate or GABA. Indeed there is evidence that glutamate receptors are expressed on both neural synaptic and glial processes
(Gallo and Ghiani 2000), and there is no way in this study of
distinguishing between glial and neural dialysate amino acids.
Nevertheless, the changes in the PAG extracellular glutamate
or GABA levels may deeply affect nociceptive perception
(Gebhart et al. 1984).
An important issue at this point regards how we have chosen
the doses of these drugs, and in particular of AMN082. The
doses of AMN082 to be administered into the PAG were
chosen based on our previous in vivo study in the mouse
(Marabese et al. 2006). Indeed, because to our knowledge no
other study has been published describing the effects of
AMN082 by using a similar administration route, we performed extensive preliminary experiments with several doses
of AMN082, as well as of (S)-3,4-DCPG, to find minimal
doses able to change RVM cell activities. In contrast to the in
vitro data showing that (S)-3,4-DCPG is expected to be more
potent than AMN082 (Mitsukawa et al. 2005; Thomas et al.
2001), we found in this study that when injected into the PAG,
2 nmol/rat of AMN082 was as effective as 4 nmol/rat of
(S)-3,4-DCPG in changing both RVM cell activities and thermoceptive thresholds. Such a discrepancy may be due to
different receptor binding sites (allosteric or orthosteric) of
these two ligands that may differ greatly in inducing receptor
internalization/desensitization and, therefore the functional responses (Ferguson 2001; Pelkey et al. 2007); the possible
different pharmacokinetics (i.e., uptake and metabolism) related to these mGluR ligands by glial and neural cells in the
whole animal. Nevertheless, it is also intriguing that MSOP
was effective at 100 nmol/rat in antagonizing the AMN082- or
the (S)-3,4-DCPG-induced effects, quite a low concentration as
compared with its in vitro IC50 (Thomas et al. 1996) that would
call for a dose 10-fold higher that used in the current study.
Indeed we cannot ignore the fact that in the whole brain glial,
endothelial, and neural processes represent functional or physical factors, which affect diffusion in the extracellular space of
drugs and also of neurotransmitters. Thus the actual drug
concentrations in the extrasynaptic compartment will be the
result of many factors contributing to the complex framework
51
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MARABESE ET AL.
matter appears to be a critical presynaptic receptor specifically
involved in the fine tuning of glutamate extracellular release.
In conclusion, this study shows that, although with different
mechanisms, mGluR7 and mGluR8 stimulation may modulate
the RVM ON- and OFF-cell activities within the PAG. Thus if on
the one hand PAG mGluR8 stimulation can play a pivotal role
in inducing analgesia (likely by inhibiting GABAergic interneurons), the stimulation of mGluR7 may, on the other hand,
reduce downstream the tonic excitatory control of glutamate on
the endogenous antinociceptive pathways originating from the
PAG. This study underlines the importance of focusing further
efforts on the investigation of mGluR8 agonist analgesic potential and on the development of mGluR7 antagonists, which
could be promising new pain-relief agents.
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Behbehani MM, Fields HL. Evidence that an excitatory connection between
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